The role of LIV-1 was assessed by its changed expression during EMT. A, ARCaPE cells were treated for 48 hours with growth factors to induce EMT. RT-PCR and Western blotting were used to show increased LIV-1 expression (left panel), and the dose responsiveness of the expression (right panel). B, Western blotting was used to confirm EMT-like expressional changes in the treated ARCaPE cells. C, mesenchymal cell-like ARCaPM cells were subjected to siRNA knockdown for LIV-1 expression for 48 hours. RT-PCR and Western blotting were used to detect expressional changes reflecting reversal of EMT in the treated cells. D, Scratch wound healing and transwell invasion assays were used to determine migratory and invasive behavior in siRNA treated ARCaPM cells. * indicates statistical significance compared to the con1 control clone (P<0.05). E, ARCaPE cells were transfected with LIV-1 expression construct. RT-PCR and western blotting were performed 48 hours after transfection to detect expressional changes reflecting EMT-like events. GAPDH served as an internal control for RT-PCR reactions, and β-actin was used as a loading control in Western blotting.

The produced antibodies to LIV-1 were subjected to validation for specificity. A, HEK293 cells transiently transfected with the LIV-1 expression construct were subjected to Western blotting analysis with the antibodies to LIV-1 (upper panel). Antibody specificity was determined by pre-absorbing the antibody with the immunizing peptide (middle panel). B, ARCaPE cells were transiently transfected with the LIV-1 expression construct to overexpress LIV-1 and ARCaPM cells with the specific siRNA to suppress LIV-1 expression. In the upper 2 panels, Western blotting was performed 48 hours later with the antibodies to LIV-1. In the lower 2 panels, these cells were examined by RT-PCR to confirm the LIV-1 expression. β-actin was used as control in Western blotting and GAPDH was used as control for RT-PCR analysis. C, IHC was conducted to further confirm LIV-1 Ab specificity in ARCaPE cells transiently transfected with the LIV-1 expression construct and ARCaPM cells transiently transfected with the specific siRNA (200 ×).

ARCaPE clones overexpressing LIV-1 displayed EMT-like changes in gene expression, cellular morphology and behavior. A, all four LIV-1 overexpressing ARCaPE clones showed EMT-like expressional changes as detected by Western blotting, while the two vector control clones (1 and 2) retained an epithelial cell-like expression profile. B, cellular morphology of the LIV-1 overexpressing cells showed marked changes from the control clones (200 ×). C, LIV-1 overexpressing cells (LIV#8 and LIV#14 were compared with vector control clones 1 and 2 and parental ARCaPE and ARCaPM cells for altered migratory capability in transwell assays. Each result is the mean ± standard deviation of a triplicate assay. D, the LIV-1 overexpressing #8 and #14 clones were compared with vector control clones 1 and 2 and parental ARCaPE and ARCaPM cells for altered invasiveness. * indicates statistical significance compared to the con1 control clone (P<0.05).

Upon subcutaneous inoculation, the two ARCaPE clones overexpressing LIV-1 (LIV8 and LIV14) were compared with vector control clones (con1 and con2) for tumor formation in athymic mice. Growth of the tumors at day 23, 32, 43, and 50 is shown. Each result represents the mean ± standard deviation of six tumors. An asterisk indicates statistical significance compared to the con1 control clone (P<0.05).

A, RT-PCR and Western blotting were used to determine LIV-1 expression in different prostate cancer cell lines. β-actin was used as control in Western blotting and GAPDH was used as control for RT-PCR analysis. B, representative IHC images showed increased LIV-1 expression in human prostate specimens from benign to bone metastasis (125 ×). C, Interval plot of LIV-1 expression is shown versus prostate cancer progression from normal/benign, PIN, primary cancer to bone metastasis. The data are shown with 95% confidence interval (n = number of cases analyzed). The median expression for LIV-1 in bone was significantly greater than in normal/benign, PIN, and primary cancer (P<0.001) and in primary cancer only (P = 0.002) as analyzed by Mann-Whitney test.

A, LIV-1 overexpressing cells (LIV#8) and control cells (con1) were treated with EGF and AG1478 for 2 hours. Western blotting showed that the EGF-elicited EGFR and ERK phosphorylation was blocked by the AG1478 inhibitor. B, RT-PCR showed increased HB-EGF, MMP2 and MMP9 expression in LIV-1 overexpressing cells. C, LIV-1 overexpressing cells (LIV#8 and LIV#14) were cultured in serum-free medium for 24 hours and the culture media were used to determine the MMP2 and MMP9 enzymatic activity by zymogram assay. D, the effect of MMP2/9 enzymatic activity on HB-EGF shedding was evaluated by ELISA. LIV-1 overexpressing clones (LIV#8 and LIV#14) secreted more HB-EGF than control clones (con1 and con2), and the secretion was reduced by MMP2/9 inhibition. ∗ indicates statistical significance compared to the control of the group (P<0.05). E, Western blotting showed that inhibition of MMP2/9 activity suppressed EGFR and the downstream ERK phosphorylation. F, LIV-1 overexpressing cells were treated with MMP 2/9 inhibitor III for 24 hours in Transwell motility assays. Both migration and invasion of the treated cells were decreased. * indicates statistical significance compared to the control group (P<0.05). G, diagram depicts the proposed role of LIV-1 in prostate cancer cell EMT and metastasis.